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, 31 (6), 2125-35

Akt Suppresses Retrograde Degeneration of Dopaminergic Axons by Inhibition of Macroautophagy

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Akt Suppresses Retrograde Degeneration of Dopaminergic Axons by Inhibition of Macroautophagy

Hsiao-Chun Cheng et al. J Neurosci.

Abstract

Axon degeneration is a hallmark of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Such degeneration is not a passive event but rather an active process mediated by mechanisms that are distinct from the canonical pathways of programmed cell death that mediate destruction of the cell soma. Little is known of the diverse mechanisms involved, particularly those of retrograde axon degeneration. We have previously observed in living animal models of degeneration in the nigrostriatal projection that a constitutively active form of the kinase, myristoylated Akt (Myr-Akt), demonstrates an ability to suppress programmed cell death and preserve the soma of dopamine neurons. Here, we show in both neurotoxin and physical injury (axotomy) models that Myr-Akt is also able to preserve dopaminergic axons due to suppression of acute retrograde axon degeneration. This cellular phenotype is associated with increased mammalian target of rapamycin (mTor) activity and can be recapitulated by a constitutively active form of the small GTPase Rheb, an upstream activator of mTor. Axon degeneration in these models is accompanied by the occurrence of macroautophagy, which is suppressed by Myr-Akt. Conditional deletion of the essential autophagy mediator Atg7 in adult mice also achieves striking axon protection in these acute models of retrograde degeneration. The protection afforded by both Myr-Akt and Atg7 deletion is robust and lasting, because it is still observed as protection of both axons and dopaminergic striatal innervation weeks after injury. We conclude that acute retrograde axon degeneration is regulated by Akt/Rheb/mTor signaling pathways.

Figures

Figure 1.
Figure 1.
Myr-Akt suppresses retrograde degeneration in dopaminergic axons. A, Confocal images taken through the MFB are shown for representative single mice treated with either a control injection of AAV DsRed or with AAV Myr-Akt, followed 3 weeks later by either ipsilateral injection of 6OHDA or axotomy. Each series of panels represents adjacent optical fields, extending from medial to lateral through the MFB, in a single section. The top group of three sets of panels represents an experiment (EXP) with 6OHDA; the bottom group of two sets of panels represents an experiment with axotomy. For comparison to lesion conditions, the contralateral, noninjected control (CON) side of a Myr-Akt mouse is shown in the middle set of panels in the top group (the noninjected sides of AAV DsRed-injected mice were comparable, as shown by axon counts in B). In the top set of panels in the top group there is a loss of axons and the appearance of axonal spheroids (red arrows) at 3 d following intrastriatal 6OHDA in the mouse treated with AAV DsRed. In the mouse treated with Myr-Akt, shown as the bottom set of panels in the top group, there is a relative preservation of axons and minimal spheroid pathology. In the bottom group of panels there is a loss of axons at 6 d following unilateral anterior MFB axotomy in a mouse treated with AAV DsRed. Relative preservation of axons is observed in the Myr-Akt condition. The central white lines in each image are separated by 10 μm. An axon was counted only if it is traversed both of these lines. B, In the top graph, MFB axons were counted in mice that had received either AAV DsRed (n = 4) or AAV Myr-Akt (n = 4) without 6OHDA lesion. Neither control AAV DsRed injection nor Myr-Akt had an effect on the number of GFP-visualized axons. In the middle graph, the effect of 6OHDA lesion is shown (6OHDA-STR, intrastriatal 6OHDA). Mice treated with AAV DsRed as a control (n = 8) demonstrated a mean loss of 37 (or 42%) of their MFB axons, whereas mice treated with Myr-Akt (n = 5) demonstrate only a mean loss of 14 axons (or 15%), a highly significant difference compared with the AAV DsRed control condition (p < 0.001, ANOVA). The 15% loss of axons in the mice treated with Myr-Akt was not significant compared with the contralateral CON noninjected side (p = 0.13, NS). In the bottom graph, the effect of unilateral anterior axotomy is shown. Mice treated with AAV DsRed (n = 9) demonstrated a mean loss of 31 (or 33%) of their axons by postlesion day 6, whereas mice treated with Myr-Akt (n = 9) showed only a mean loss of 4 (or 4%) of their axons, a highly significant difference (p < 0.001, ANOVA). The 4% loss of axons in the mice treated with Myr-Akt was not significant compared with contralateral CON nonaxotomized MFB (p = 0.7, NS). C, Myr-Akt provides a robust lasting protection of dopaminergic striatal terminals following intrastriatal 6OHDA. At 4 weeks following lesion, mice were processed for immunostaining of DAT to assess striatal dopaminergic innervation. Representative coronal sections reveal that DAT immunostaining is eliminated following unilateral 6OHDA in AAV GFP (control)-injected mice, whereas there remains a moderate degree of preservation, particularly in the ventromedial quadrant in mouse treated with Myr-Akt. This result is shown quantitatively as optical density of staining on the lesioned side as a percentage of the contralateral, nonlesioned side. Among mice treated with AAV GFP, optical density was reduced to 11% of control, whereas among AAV Myr-Akt-treated mice 22% of staining remained (p = 0.04, t test; AAV GFP, n = 5; Myr-Akt, n = 7).
Figure 2.
Figure 2.
Ultrastructural features of autophagy in striatal neuropil following 6OHDA injection. A, A single autophagic vacuole, AV, is observed in a striatal neurite 1 day following intrastriatal 6OHDA. The AV is defined by a characteristic double membrane (DM), and it contains a degenerating mitochondrion (MITO). To assist the visual presentation, the cytoplasm of the cell has been colorized in semitransparent pink, and adjacent extracellular space (ECS) is in blue. Only the definitively clear ECS adjacent to the profile of interest has been colorized. In the right panel, the cellular features of the boxed area in the left panel are shown in detail. ECS (blue arrow) separates the plasma membrane of an adjacent cell (PM1) from the plasma membrane (PM2) of the AV-containing cell. A thin rim of cytoplasm (pink) separates PM2 from the outer double membrane of the AV (AV M1) for most of its extent, except at one region where AV M1 abuts the inner surface of PM2 (*, white bracket). The inner membrane of the AV double membrane (AV M2) runs parallel to AV M1. B, An AV is observed in a neurite following injection of 6OHDA into the MFB. A characteristic double membrane (DM) is again observed. The right panel, an enlarged view of the boxed area in the left panel, reveals that the outermost membrane of the AV (AV M1) is adjacent to two synaptic vesicles (SV) in the cytoplasm of the neurite. This AV is multilamellar, with four concentric membranes (AV M1–4) indicated by white arrows in the right panel. The formation of multiple concentric lamellae is highly characteristic of AVs (Hornung et al., 1989; Jia et al., 1997; Hariri et al., 2000; Nixon et al., 2005). C, An AV is observed in striatal neuropil following injection of 6OHDA into the MFB. This AV contains several multilamellar bodies (MLB). The right panel, an enlarged view of the boxed area in the left panel, shows that ECS separates the plasma membrane (PM) of the AV-containing cell from the external myelin layer of a myelinated axon. A thin rim of cytoplasm (C, colored pink) separates the membrane of the AV (AVM) from the PM. A single MLB is subjacent to the outermost AV membrane (white arrow).
Figure 3.
Figure 3.
Autophagy occurs in dopamine neuron cell bodies and axons in diverse models of axon injury. A, The presence of AVs in dopaminergic axons and cell bodies is identified in the intrastriatal 6OHDA and in both the anterior axotomy (AXOT ANT) and posterior axotomy (AXOT POST) models by DsRed-LC3 labeling in TH-GFP mice. In the top panels, two clusters of AVs (red arrows) are identified in a GFP-labeled dopaminergic axon in the MFB 2 d following intrastriatal 6OHDA (6OHDA STR). Localization of the AVs within the axon was confirmed by virtual rotation of the set of Z-stack images (see Movie S1, available at www.jneurosci.org as supplemental material). AVs are also observed in dopaminergic cell bodes in the SNpc. A single example is identified by a red arrow. In the bottom two sets of panels, red punctuate AVs are identified in dopaminergic neurons of the SNpc following both anterior and posterior axotomy. Single examples are indicated by red arrows. Bar,10 μm. ANT, Anterograde; RET, retrograde. B, To monitor the number of AVs following lesions, mice were preinjected into the SN with AAV GFP-LC3 in a 1:1 mixture with either AAV Myr-Akt or AAV DsRed as control. After 3 weeks, all mice received a unilateral intrastriatal 6OHDA injection and were then killed at the indicated postlesion day (PLD) to quantify AVs in SNpc neurons as green GFP-LC3-positive puncta. The top graph shows that Myr-Akt decreased the number of AVs at all PLDs. On PLD 1, Myr-Akt reduced the number of neurons with AVs by 79% (p < 0.001, n = 6 both groups). The middle graph shows similar results when expressed as the total number of AVs in each SN section. To monitor the number of AVs in dopamine neurons following anterior axotomy, we used DsRed-LC3 in TH-GFP mice as illustrated in A. The reason for this change in methodology was that the axotomy lesion, unlike the 6OHDA lesion, is not selective for dopamine neurons, and our experimental goal was to monitor AV number specifically in this population. Therefore, TH-GFP mice were preinjected into the SN with AAV DsRed-LC3 in a 1:1 mixture with Myr-Akt or DsRed-LC3 alone. After 3 weeks, all mice received a unilateral anterior axotomy and were killed at 2 d postlesion to quantify the number of GFP-positive SNpc neurons with discrete red puncta, as shown in A. Myr-Akt reduced the number of neurons with AVs by 78% (p < 0.001, n = 6 both groups).
Figure 4.
Figure 4.
Myr-Akt mediates axon protection through mTor signaling. A, Akt regulates mTor signaling by phosphorylation and inhibition of the tuberous sclerosis complex 2 (TSC2) GTPase-activating protein for Rheb-GTPase. Released from inhibition by TSC, Rheb-GTPase activates mTORC1, which phosphorylates downstream targets such as 4E-BP1. In addition, mTor signaling negatively regulates autophagy. B, Western analysis of phospho-mTor(Ser2448) in ventral mesencephalon whole tissue extracts 3 weeks following unilateral AAV SN injection shows an increase in abundance of phosphorylated mTor protein on the side of AAV injection [E (blots) and EXP (graph), Experimental] compared with the noninjected contralateral control [C (blots) and CON (graphs)] for AAV Myr-Akt, but not AAV DsRed. The increase induced by Myr-Akt is shown quantitatively in the bar graph depicting the optical density of p-mTor bands normalized for the optical density of the corresponding β-actin band (n = 3). C, Immunoperoxidase staining for p-4E-BP1 is observed in neurons in the SNpc (arrows) following transduction with AAV Myr-Akt on the injected side, but not on the contralateral control side. At a cellular level, positive staining for p-4E-BP1 is observed exclusively in neurons expressing Myr-Akt, identified by positive FLAG staining. A total of 50 p-4E-BP1-positive neurons were identified, and all were also positive for FLAG. Among p-4E-BP1-positive neurons, there was a 1.7-fold increase in cross-sectional area (p < 0.001, n = 25 neurons in each group). At a subcellular level, p-4E-BP1 expression colocalizes with that of cathepsin-D, a lysosomal marker. The white rectangles in the lower magnification images are shown at higher magnification in the right-hand panels. D, Both Myr-Akt-FLAG and p-4E-BP1 are expressed in axons in the MFB and the striatum. E, To assess the role of mTor signaling in the ability of Myr-Akt to protect axons, we created an AAV vector expressing a constitutively active form of human Rheb, hRheb(S16H). Transduction of the SNpc is demonstrated by immunoperoxidase staining for FLAG in the top panel. Successful transduction of dopamine neurons (with an efficiency ranging from 80 to 90%) is demonstrated by fluorescent double-labeling for TH and FLAG. As for AAV Myr-Akt, transduction with AAV hRheb(S16H) induces an abundant increase in the number of p-4E-BP1-positive neurons, demonstrated by immunoperoxidase staining. F, Following transduction with AAV hRheb(S16H), axons of dopaminergic neurons are resistant to retrograde degeneration induced by intrastriatal 6OHDA (6OHDA-STR). A mean loss of 26 axons (or 36%) occurred in the mice treated with DsRed (n = 6), whereas a mean loss of only 12 axons (or 16%) occurred in the mice treated with hRheb(S16H) (n = 7; p < 0.001, ANOVA). The 16% loss in the MFB following 6OHDA in the hRheb(S16H)-treated mice was not significant (p = 0.09, NS).
Figure 5.
Figure 5.
Following deletion of Atg7, axons of SNpc dopamine neurons are resistant to retrograde axon degeneration. A, In the absence of AAV Cre injection, Atg7fl/fl:TH-GFP mice show a loss of MFB dopaminergic axons (visualized by confocal microscopy of endogenous GFP) and the appearance of axonal spheroid pathology (red arrows) following unilateral 6OHDA injection. Atg7w/w:TH-GFP mice injected with AAV Cre show a similar axon loss and pathology. However, following injection of AAV Cre, Atg7fl/fl:TH-GFP mice show minimal axon loss and pathology following 6OHDA injection. Following 6OHDA injection, Atg7fl/fl:TH-GFP mice without AAV Cre (n = 5) and Atg7w/w:TH-GFP mice given AAV Cre (n = 6) show a mean loss of 31 (or 32%) and 32 (or 34%) MFB axons respectively, whereas Atg7fl/fl:TH-GFP mice treated with AAV Cre (n = 6) show a mean loss of only 5 (or 5%) (p < 0.001, ANOVA). The minor loss of axons (5%) on the 6OHDA-treated side is not significant compared with the contralateral, noninjected control (CON) side (p = 0.7). 6OHDA-STR, Intrastriatal 6OHD. B, Following deletion of Atg7, nigrostriatal axons in Atg7fl/fl:TH-GFP mice show less pathology and relatively preserved number following anterior MFB axotomy (AXOT) compared with Atg7wt/wt:TH-GFP mice. In the Atg7wt/wt:TH-GFP mice, numerous axon spheroids are observed (red arrows). These mice (n = 6) demonstrated a mean loss of 53% of their axons by PLD 6, whereas Atg7fl/fl:TH-GFP mice injected with AAV Cre (n = 4) show only a 26% loss, a highly significant difference (p < 0.001, ANOVA). C, Abrogation of autophagy by deletion of Atg7 provides a robust and lasting neuroprotection of axons following intrastriatal 6OHDA lesion. At 4 weeks following lesion, Atg7wt/wt:TH-GFP mice (n = 3) demonstrate a mean loss of 78% of their axons, whereas Atg7fl/fl:TH-GFP mice injected with AAV Cre (n = 3) reveal still only a 34% loss (p < 0.001, ANOVA). FL or fl, Flox; WT or wt, wild type; EXP, experimental.

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